Low temperature injury of higher plants is largely categorized into two different types. One is the injury caused by temperatures at or below 0.degree. C. and is called "freezing injury". The other, which is the subject matter of the present invention, is called "chilling injury" and is totally different from freezing injury. Most tropical and subtropical plants suffer chilling injury at temperatures in the range of 5.degree. to 15.degree. C., which injury damages the tissue(s) of whole and/or a part of the plants leading to a variety of physiological dysfunctions and ultimately to death in the severest cases.
Plants susceptible to chilling injury are called "chilling-sensitive" plants and include many important crops such as rice, maize, yam, sweet potato, cucumber, green pepper, eggplant, squash, banana, melon, kalanchoe, cyclamen, lily, rose, castor bean, sponge cucumber and tobacco. These plants suffer a variety of injuries, such as the inhibition of germination and growth, tissue necrosis as well as the death of the whole plant, at temperatures between 5.degree. and 15.degree. C., in most cases at around 10.degree. C., and thus are prone to damage by cold weather and frost. Furthermore, fruits, vegetables, and the like harvested from chilling-sensitive plants cannot tolerate low temperature storage (as illustrated by the black decaying spots that quickly appear on bananas when taken out of a refrigerator) making it difficult to store these harvests for a long period after the harvest.
Most plants of temperate origin, on the other hand, are chilling-resistant and are not injured even by a low temperature of around 0.degree. C. Chilling-resistant crop plants include wheat, barley, spinach, lettuce, radish, pea, leek, and cabbage. Wild weeds such as dandelion and Arabidopsis are also chilling-resistant.
Chilling injury is significantly related to the fluidity of membrane lipids that constitute biomembranes. Biomembranes are one of basic organizing units of living cells. They define the inside and outside of cells as the cell membrane and in eukaryotic cells also organize a variety of membrane structures (cell organelles) to partition the cell into several functional units. Biomembranes are not mere physical barriers against high molecular weight substances and low molecular weight electrolytes; the function of proteins associated with the membranes allow the selective permeation, and/or the active transport against concentration gradient, of particular substances. In this way biomembranes keep the micro-environment of cytoplasm and cell organelles in a suitable condition for their purpose. Some biochemical processes, such as energy production by respiration and photosynthesis, require a specific concentration gradient of particular substances across biomembranes. In photosynthesis, the energy of light generates a hydrogen ion gradient potential across the thylakoid membrane within chloroplasts, which potential energy is then convened to ATP, a high-energy compound utilized by living cells, by proteins in the thylakoid membrane. Accordingly, if biomembranes fail to function as a barrier as described above, it will disturb not only the micro-environment of cells but impair these cellular functions based on a concentration gradient, leading to serious dysfunctions of living cells.
The membrane lipids that constitute biomembranes are mainly phospholipids and, in the case of chloroplasts, glycerolipids. Phospholipids are 1,2-di long-chain alkyl (fatty acyl) esters of glycerol with a polar group bonded at the 3 position as a phosphoester. They are amphipathic compounds having both a hydrophilic portion (the polar group) and a hydrophobic portion (the fatty acyl groups) within one molecule and therefore form a lipid bilayer with the hydrophobic portions inside and the hydrophilic portions on the surface when dispersed in an aqueous solution. This lipid bilayer is the basic structure of biomembranes which "buries" a variety of proteins inside and/or on its surface. Under physiological conditions, the lipid bilayer is in the liquid-crystalline phase in which the inside of the bilayer retains a high fluidity, allowing free horizontal dispersion and rotation of protein and lipid molecules within the membrane. This fluidity of biomembranes is essential for cellular functions (Darnell, J. et al, Molecular cell biology, Scientific American Books, 1986).
When the temperature of a simple lipid bilayer in the liquid-crystalline phase is lowered to a certain temperature called the phase transition temperature (Tc), the bilayer undergoes a phase transition to the gel phase in which the inside of the membrane has less fluidity. In the case of biomembranes, which consist of different types of lipids, some lipids with a high Tc begin to form gel phase domains at a certain temperature while other lipids with a lower Tc are still in the liquid-crystalline phase, resulting in the phase separation, in which both the liquid-crystalline and gel phases co-exist. In a phase separated state, biomembranes become leaky and no more serve as a barrier against low molecular weight electrolytes.
A relationship between chilling injury and the phase transition of membrane lipids was first proposed in early 1970's (Lyons, J. M., Ann. Rev. Plant Physiol., 24:445, 1973). At that time, however, there was no concrete data supporting the existence of the relationship. Later, in a series of experiments using cyanobacteria (blue-green algae) as model organisms, it was shown that the chilling injury of cyanobacteria is the result of irreversible effluent from the cells of electrolytes such as ions following the phase separation of the cell membrane at a chilling temperature (Murata, N. and Nishida, I., in The biochemistry of plants vol. 9 Lipids: Structure and function, p.315, Academic Press, Orlando, 1987).
Lipids are generally classified by the polar group (see above for the structure of membrane lipids), since their behavior in column and thin layer chromatographics is largely determined by the polar group. Among one particular class of lipids with the same polar group, there are many different molecules with various combinations of the two fatty acyl groups in the molecule. The term "molecular species" is used to distinguish these molecules. The Tc of each lipid molecular species depends on the polar group as well as the chain length and degree of unsaturation (the number of double bonds) of the fatty acyl groups, and in some instances the environmental salt concentration and such. Among these, the degree of unsaturation of the fatty acyl groups has the largest effect; while a particular molecular species with two saturated fatty acyl groups usually has a Tc above room temperature, introduction of only one double bond into one of the fatty acyl groups results in the decrease of Tc to around 0.degree. C. (Santaten, J. F. et al, Biochem. Biophys. Acta, 687:231, 1982). (However, if the double bond is in the trans configuration, the effect on the Tc is very small [Phillips, M. C. et al, Chem. Phys. Lipids, 8:127, 1972]. Most double bonds of membrane lipids are in the cis configuration and the trans configuration is relatively rare.) This indicates that a lipid molecular species with at least one double bond in its fatty acyl groups (hereinafter called "unsaturated molecular species") does not undergo phase transition at around 10.degree. C., the critical temperature for chilling injury. Consequently, only those lipid molecules with two saturated fatty acyl groups (hereinafter called "saturated molecular species") could induce the phase separation of biomembranes which is considered to be the primary event in chilling injury.
Membrane lipids have been extracted from several chilling-sensitive and resistant plants, separated according to the polar group, and their fatty acid and molecular species compositions analyzed. The results showed that only phosphatidylglycerol (PG) contains a significant amount of saturated molecular species among plant membrane lipids and that the content of saturated molecules in PG is high (30-70 %) in chilling-sensitive plants and low (&lt;20 %) in chilling-resistant plants (Murata, N., Plant Cell Physiol, 24:81, 1983; Roughan, P. G., Plant Physiol., 77:740, 1985). Since PG is a major component of plastid (chloroplast, chromoplast) biomembranes, this correlation between the PG molecular species composition and chilling sensitivity strongly suggests that the primary event in the chilling injury of higher plants is the phase separation of plastid biomembranes induced by the phase transition of PG (Murata, N. and Nishida, I., in Chilling injury of horticultural crops, p.181, CRC Press, Boca Raton, 1990).
PG is localized in plastids and, in the case of green leaves, synthesized mainly in chloroplasts (Sparace, S. A. and Mudd, J. B., Plant Physiol., 70:1260, 1982). Its biosynthesis follows the steps shown below.
1. Transfer of a fatty acyl group to the sn-1 position of glycerol 3-phosphate. PA1 2. Transfer of another fatty acyl group to the sn-2 position. PA1 3. Esterification of glycerol to the 3-phosphate group. PA1 4. Desaturation of fatty acyl groups on the molecule.
Fatty acids are exclusively synthesized in chloroplasts. The synthesized fatty acids are supplied to steps 1 and 2 of PG synthesis as acyl-ACP complexes wherein the fatty acids are bound to a protein called acyl carrier protein (ACP). Most of the fatty acids synthesized in chloroplasts are palmitic acid (saturated C-16 acid, hereinafter designated as 16:0) and oleic acid (mono-unsaturated C-18 acid, hereinafter designated as 18:1).
Step 1 of the above scheme is catalyzed by acyl-ACP:glycerol 3-phosphate acyltransferase (EC 2.3.1.15) (hereinafter called ATase). This enzyme is a soluble enzyme in chloroplast stroma. It has been partially purified from spinach and pea (Bertrams, M. and Heinz, E., Plant Physiol., 68:653, 1981) and purified to homogeneity from squash (Nishida, I. et al, Plant Cell Physiol., 28:1071, 1987). It is encoded by a nuclear gene, which has been cloned from squash, Arabidopsis and recently from pea (Ishizaki, O. et al, FEBS Lett., 238:424, 1988; Nishida, I. et al., in Plant lipid biochemistry, structure and utilization, Portland Press, London, 1990; Weber, S. et al, Plant Molec. Biol., 17:1067, 1991). ATases from different sources differ in selectivity for the substrate, acyl-ACP. While ATascs from spinach, pea and Arabidopsis, which arc chilling-resistant, have a high selectivity for 18:1-ACP, ATase from squash, a chilling-sensitive plant, equally utilizes both 18:1-ACP and 16:0-ACP (Frentzen, M. et al, Eur. J. Biochem., 129:629, 1983; Frentzen, M. et al., Plant Cell Physiol, 28:1195, 1988 ).
The enzyme that catalyzes step 2 of the above scheme is a membrane-bound enzyme of chloroplast envelope and utilizes only 16:0-ACP (Frentzen, M. et al, Eur. J. Biochem., 129:629, 1983). In a number of plant species called 16:3 plants, the intermediate product of steps 1 and 2, phosphatidic acid (1,2-diacylglyccrol 3-phosphate), is also an intermediate compound for the biosynthesis of glycerolipids (mono- and digalactosyldiacylglyccrols and sulfoquinovosyl-diacylglyccrol) synthesized in chloroplasts. Steps 1 and 2 are therefore common to the lipid biosynthesis in chloroplasts of the 16:3 plants.
Very little is known about the enzymes for steps 3 and 4 of PG biosynthesis. However, it is well known that the desaturation of fatty acyl groups in PG is asymmetric. At the sn-1 position, most of 18:1 is further desaturated to have two or three double bonds while 16:0 is not desaturated. At the sn-2 position, some of the bound 16:0 is desaturated to 3-trans-hexadecenoic acid (hereinafter designated as 16:1t) but no cis-double bond is introduced. Since a trans-double bond is much less effective in decreasing the phase transition temperature, the conversion of 16:0 to 16:1t at tic position 2 of PG would decrease the Tc by only about 10.degree. C., so that the Tc is still higher than the critical temperature for chilling injury (Bishop, D. G. and Kenrick, J. R., Phytochemistry, 26:3065, 1987). PG molecular species with 16:1t are accordingly included within saturated molecular species hereinafter. Because no cis-double bond is introduced in the fatty acyl group at position 2, the fatty acyl group at the position 1 is very important in determining the content of saturated molecular species.
Chilling-sensitive crop plants suffer significant disadvantages in low-temperature tolerance and long-term post-harvest storage as described above. Nevertheless, many of chilling-sensitive crops are very important and indispensable for agricultural production; for example, rice and maize are the main cereal crops in many parts of the world. An improvement in the chilling resistance of these crops would make it easier to grow them in a chilling environment and/or to store their harvest for a long period. In the case of ornamental flowers and vegetables grown in a greenhouse due to their chilling-sensitivity, improvement of chilling resistance would make the greenhouse unnecessary or save the heating expense to a great extent. Furthermore, the improvement might expand the area where the crop is grown, since temperature is often the main factor to define the borders of crop development.
There is thus a significant demand for chilling resistant plants and chilling resistance has been one of the major goals of crop breeding. However, conventional crossing breeding is limited in genetic sources for this purpose, because one can cross the crop only within the same species. Recent progress in genetic engineering of higher plants has made it possible to introduce genetic information into crops from an unlimited range of genetic sources. The application of genetic engineering to providing chilling-resistance would therefore be invaluable.
As already described, the primary event in the chilling injury of higher plants is the phase separation of plastidial membranes, and the plastidial membranes of chilling-sensitive plants contain a higher mount of the saturated PG molecular species considered to induce the phase separation. It was thus suggested that it might be possible to increase the chilling resistance of chilling-sensitive plants by changing the fatty acid composition of their PG to decrease the content of saturated molecular species (Murata, N., Plant Cell Physiol, 24:81, 1983). However, this was only a hypothesis and, to date, there has been no report of any method to change the fatty acid composition of cellular lipids nor any report of a plant with an altered fatty acid composition.